LIN28B / Protein lin-28 homolog B · Western blot design guide

Design a Western Blot for LIN28B

Real validated LIN28B Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-LIN28B WB antibodies. Everything you need to plan the experiment before you commit precious samples.

Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use

Western blot protocol sheet for LIN28B: expected band ~27.1 kDa, hero antibody A00896-1, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **LIN28B** Western blot protocol sheet — expected band ~27.1 kDa, antibody A00896-1, controls and PMC citations. Open the full LIN28B WB guide →

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LIN28B Western Blot Experimental Design Guide

Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.

Must know before running

	Expected band	~27.1 kDa
	Observed band	~35 kDa
	Gel	5–20% (catalog A00896-1)
	Negative control ⓘ	Suggested KO / knockdown lysate

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	Phosphorylated
	Caveat	Phosphorylation-state controls
	Gene-set association	MSigDB C7 membership
	Isoform	2 isoform(s)

Section 1

Real Curated LIN28B Western Blot Protocols

The A00896-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.

Recommended Western blot protocol parameters
Sample / lysate	human 293T, human HepG2 (catalog A00896-1)
Gel %	5–20% (catalog A00896-1)
Load	30 ug; reducing conditions (catalog A00896-1)
Transfer	a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00896-1)
Membrane	nitrocellulose membrane (catalog A00896-1)
Blocking	5% non-fat milk/TBS for 1.5 hour at RT (catalog A00896-1)
Primary antibody	A00896-1 · 0.5 μg/mL (catalog A00896-1)
Primary incubation	overnight at 4°C (catalog A00896-1)
Secondary antibody	goat anti-rabbit IgG-HRP, 1:5000 (catalog A00896-1)
Secondary incubation	1.5 hour at RT (catalog A00896-1)
Wash	TBS-0.1%Tween 3 times with 5 minutes each (catalog A00896-1)
Detection	ECL (catalog A00896-1)

Section 2

What Is the Expected LIN28B Western Blot Band Size?

LIN28B is predicted at 27.1 kDa and observed near 35 kDa in whole-cell blots; the cause of the difference is not established.

What am I looking at on my blot?
Band near 35 kDa	Empirical LIN28B band in reducing whole-cell blots; confirm identity with controls
Band near 27 kDa	Near the predicted mass, but identity requires confirmation
Additional bands at different positions	Isoforms 1 and 2 are possible contributors; distinct migration is unestablished
Faint cytoplasmic fraction band	Consistent with predominantly nucleolar localization, depending on cell type

💡Expected LIN28B appearanceLIN28B is predicted at 27.1 kDa, while reducing whole-cell blots show a band near 35 kDa; confirm band identity with appropriate controls because the reason for the difference is unestablished.

How each factor affects band size
Predicted molecular mass	27.1 kDa calculated mass versus the observed band near 35 kDa; cause unresolved
250-residue sequence	Underlies the calculated full-length mass of 27.1 kDa
Splice isoform 1	May differ in size from isoform 2; its individual mass and migration are unknown
Splice isoform 2	May differ in size from isoform 1; its individual mass and migration are unknown

Why is my band missing or off?
Situation	Likely cause	Next action
Band higher than expected	The 35 kDa observed band exceeds the 27.1 kDa prediction for an unestablished reason	Compare with the reported 35 kDa band and verify identity using an independent antibody or LIN28B depletion
Band lower than expected	An isoform could contribute, but its size is unknown	Check antibody epitope coverage and verify the band by LIN28B depletion
Multiple bands	Isoforms 1 and 2 are annotated, but distinct bands are unproven	Compare isoform-specific expression and confirm each band by LIN28B depletion
Weak or no signal	Predominantly nucleolar localization may limit signal in cytoplasmic fractions	Examine a nuclear fraction or whole-cell lysate alongside a positive control
Fragments below expected size	Sample degradation or nonspecific antibody binding; no cleavage feature is annotated	Prepare fresh lysate with protease inhibitors and verify fragments by LIN28B depletion

Source: UniProt Q6ZN17 Human Protein Atlas product datasheet (A00896-1)

Experiment evidence

Western blot images showing LIN28B band

A00896-1 · LIN28B

M00896 · LIN28B

← swipe / scroll for more images →

Real WB data behind the band-size expectations in this section.

Sample controls for LIN28B Western blot

🧪For positive controls for LIN28B in Western blot, you can use no HPA-supported positive sample from the supplied evidence.

Positive control: No high/medium HPA tissue identified

Negative control: Suggested KO / knockdown lysate

Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.

⚠️Feasibility: HPA lists no positive candidate, so a positive control cannot be selected from this evidence.

HPA tissue expression evidence for LIN28B

Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.

Higher expression tissues · candidate positive controls from IHC

Tissue	Cell type	Level	Evidence	Source
No high/medium HPA tissues identified in the supplied evidence.

Lower expression tissues · IHC evidence, not confirmed WB-negative controls

Tissue	Cell type	Level	Evidence	Source
No lower-expression tissue rows available in the supplied evidence.

Section 3

Advanced LIN28B Western Blot Tips

Deeper troubleshooting and optimisation questions for LIN28B, answered from its protein features.

How should LIN28B band migration be interpreted?

Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.

How could LIN28B isoforms affect band interpretation?

Isoforms · UniProt lists isoforms 1 and 2; isoform 2 lacks canonical residues 1–70. An antibody recognizing that region may miss isoform 2. Check the antibody epitope before assigning bands to either isoform.

Which LIN28B phosphorylation sites matter when assessing bands?

PTM · UniProt lists phosphoserines at canonical positions 54, 96, 105, 110, and 203. Canonical Ser54 is absent from isoform 2 because it lacks residues 1–70. These sites establish phosphorylation as a possibility, but do not establish a visible shift or explain the approximately 35 kDa band.

Does this guide establish induction of LIN28B?

Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.

What transfer method to use for LIN28B Western blot?

Transfer · Plan transfer for a protein with a predicted mass of 27.1 kDa and an observed band near 35 kDa. A 0.2 µm membrane can help retain proteins in this size range; check both membrane and post-transfer gel to assess retention and transfer efficiency.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the A00896-1 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.

How should LIN28B band intensity be quantified?

Quantitation · Quantify the same identified band across comparable samples and cellular fractions, using a suitable loading reference. Because UniProt lists two isoforms and multiple cellular locations, avoid combining distinct bands or fractions into one LIN28B measurement without establishing what they represent.

Why might LIN28B appear near 35 kDa instead of 27.1 kDa?

Interpretation · The supplied Western blot observation is approximately 35 kDa, while the predicted mass is 27.1 kDa. Treat 35 kDa as an empirical reference, but do not attribute the difference to a specific modification or isoform without additional evidence.

UniProt places LIN28B in the nucleus, nucleolus, and cytoplasm, with predominantly nucleolar localization noted. Consider whole-cell lysate or the relevant cellular fraction, and compare like fractions when assessing band intensity.

First compare them with the approximately 35 kDa observed band and check whether the antibody recognizes the region missing from isoform 2 (canonical residues 1–70). UniProt also lists phosphoserines, but their presence alone cannot identify an unexpected band. Verify band identity before assigning it to an isoform or modification.

Boster reagents

LIN28B Western Blot Antibodies

Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.

Real WB data

Western blot analysis of LIN28B using anti-LIN28B antibody (A00896-1). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human 293T whole cell lysates, Lane 2: human HepG2 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-LIN28B antigen affinity purified polyclonal antibody (Catalog # A00896-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for LIN28B at approximately 35 kDa. The expected band size for LIN28B is at 27 kDa.

Anti-LIN28B Antibody Picoband®

Cat # A00896-1

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Real WB data

Western blot analysis of LIN28B using anti-LIN28B antibody (M00896). <br>
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. <br>
Lane 1: human K562 whole cell lysates,<br>
Lane 2: human 293T whole cell lysates,<br>
Lane 3: human HepG2 whole cell lysates,<br>
Lane 4: human HUH-7 whole cell lysates,<br>
Lane 5: human HEL whole cell lysates.<br>
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-LIN28B antigen affinity purified monoclonal antibody (Catalog # M00896) at 1:500 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:500 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for LIN28B at approximately 35 kDa. The expected band size for LIN28B is at 27 kDa.

Anti-Lin28B Rabbit Monoclonal Antibody

Cat # M00896

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Two human-reactive anti-LIN28B antibodies have supplied WB images using human whole-cell lysates. Both captions report a band near 35 kDa, compared with an expected 27 kDa. The supplied evidence does not explain this difference.

Which to pick: Both A00896-1 and M00896 have WB images with 293T and HepG2 lysates. Choose M00896 if its additional K562, HUH-7, or HEL examples better match your sample; otherwise, choose based on the listed polyclonal or monoclonal format and assay conditions.

Source: BosterBio LIN28B gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry Q6ZN17.
Human Protein Atlas. LIN28B tissue expression.
PMC5920276 — target-verified WB comparison
PMC8382182 — target-verified WB comparison
PMC9792516 — target-verified WB comparison